Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
1. CARDIOVASCULAR DISEASE
The two most prevalent causes of death in Western civilization, myocardial and cerebral infarctions, are primarily the result of atherosclerotic lesions. Atherosclerosis is a disease of artery walls, which typically affects the major arteries supplying the heart and brain with oxygen and nutrients. It causes a thickening of artery walls which can lead to restricted blood flow, thrombosis, and eventually myocardial infarction or stroke.
Angina is a common manifestation of the disease when it affects the vessels supplying the heart, and is caused by an imbalance in the cardiac blood supply and demand for oxygen, and vasoconstriction of the affected arteries.
Positive risk factors for the development of atherosclerosis, myocardial infarction and stroke include high serum cholesterol (in low density lipoprotein (LDL) or very low density lipoprotein (VLDL) particles), triglycerides, lipoprotein (a), or fibrinogen levels, hypertension, diabetes, smoking, age and a family history of cardiovascular disease. Negative risk factors include high density lipoprotein (HDL) cholesterol or apolipoprotein AI levels.
In advanced cases of the disease, the area of artery thickening, termed the atherosclerotic plaque, is characterized by a fibrous cap rich in connective tissue, a necrotic cholesterol-rich core, hyperproliferation of smooth muscle cells and macrophages, ulceration, intraplaque hemorrhage and thrombosis, and calcification.
The potential benefit from having effective inhibitors of atherosclerosis is enormous. About 900,000 people per year in the USA die from diseases caused by atherosclerosis (i.e. myocardial infarction, stroke), more than that from all cancers combined. The costs to society of this disease are estimated to be about $84 billion per year in the U.S.A. alone. Furthermore, in many parts of Europe the incidence of atherosclerosis-related disease is considerably higher than in the USA.
2. ATHEROSCLEROSIS
Atherosclerotic lesions are typically characterized by the rapid local proliferation of vascular smooth muscle cells, deposition of intra- and extracellular lipid, and the accumulation of extracellular matrix components. Current evidence, largely from animal models and cellular studies, suggests the following pathway for atherogenesis; hypercholesterolemia is thought to result in increased levels of oxidized lipoproteins (e.g. oxidized LDL, possibly formed as a result of 15-lipoxygenase activity, (1), which can lead to abnormal localized expression of endothelial-monocyte cell adhesion molecules, and monocyte attachment to the endothelium and entry into the arterial wall (2, 3). These monocytes differentiate to macrophages and accumulate large quantities of cholesterol esters to become `foam cells` (4). It is these cells which predominate in the `fatty streak` lesion characteristic of this stage. Macrophages, vascular smooth muscle cells (SMCs), endothelial cells, and probably at a later stage adhering platelets, are thought to release growth and chemotactic factors which stimulate the uncontrolled proliferation, differentiation or migration of cells at the site of the lesion, particularly SMCs (4). bFGF (5), interleukin-1 (IL-1) (6), PDGF (7), colony-stimulating factor-1 (CSF-1), G-CSF, GM-CSF, monocyte chemoattractant protein-1 (MCP-1, (3, 8) and other factors have all been implicated in this process, and it is possible that many of these could play a role during the process of atherogenesis, either directly or indirectly, through independent actions or synergistically, possibly at different times during plaque development, or on different cell types. Many of these factors and their receptors have been directly measured in atherosclerotic tissue and found to be present at elevated levels compared to healthy arterial tissue, e.g. PDGF-B, PDGF .beta.-receptors (9, 10). Several of these factors are released from macrophages or endothelial cells in response to oxidized LDL, cytokines e.g. IL-1, CSF-1 (3), G-CSF (8, 11), GM-CSF, MCP-1. Early progenitor cells of the macrophage lineage have been found in atherosclerotic plaques (12), and their stimulation by these CSFs may be at least partially responsible for the accumulation of large numbers of macrophages at plaques.
In human atherogenesis the situation may be slightly more complicated. Here at least two types of early lesions can be identified: fatty streak lesions, characterized by numerous fat filled cells, and focal intimal thickening, characterized by edema, smooth muscle cell proliferation and little or no accumulation of lipid (13). These two lesion types can occur quite independently or together, as observed in the lower abdominal aorta. In addition, fatty deposits, although common in Western society, may be missing in populations where other risk factors such as hypertension, cigarette smoking, and diabetes are associated with increased incidence of the disease. In all situations, however, it is clear that uncontrolled cellular proliferation is a major factor in advanced plaque formation, regardless of the initiating event(s), and that control of this proliferation by pharmacological intervention should be beneficial in controlling or preventing plaque formation.
In the United States, it is estimated that 10 million people suffer from diabetes mellitus, 60 million from hypertension, and 3 million from the combination of the two. Diabetic hypertension is associated with non insulin-dependent diabetes mellitus (NIDDM) or type II diabetes. Other cardiovascular diseases, especially atherosclerosis are also associated with diabetes. It is thought that the control of hyperglycemia might help the cardiovascular pathology associated with diabetes. Reducing gluconeogenesis is one way of reducing hyperglycemia. Insulin regulates the amount of serum glucose by promoting clearance in the skeletal muscle and at the same time preventing glucose production by inhibiting hepatic gluconeogenesis and glycogenolysis. The rate limiting step of gluconeogenisis is catalyzed by the enzyme phosphoenolpyruvate carboxykinase (PEPCK) which converts pyruvate to oxaloacetic acid. PEPCK levels are determined by insulin control by insulin. In the NIDDM condition, even hyperinsulinemia fails to inhibit PEPCK transcription leading to hyperglycemia. Therefore, pharmacological downregulation of PEPCK transcription is of potential therapeutic value for the clinical management of diabetic hypertension or for other cardiovascular diabetic diseases.
3. CHOLESTEROL AND HYPOCHOLESTEROLEMIC DRUGS
One of the leading risk factors for atherosclerosis is chronic hypercholesterolemia. A causal link between hypercholesterolemia due to elevated plasma concentrations of low-density lipoproteins (LDL) and very-low-density lipoprotein (VLDL) remnants and the premature development of atherosclerosis in humans is well established (14). Patients with hereditary disorders such as familial hypercholesterolemia are predisposed towards premature coronary artery disease. Long-term effective hypolipidemic therapy aimed at reducing plasma concentrations of atherogenic lipoproteins can prove beneficial not only for these patients but also for those with persistent hypercholesterolemia attributable to secondary disorders such as the nephrotic syndrome. Lowering of plasma concentration of both total and low-density lipoprotein, either by diet or drug therapy, has provided conclusive evidence that hypocholesterolemic therapy given to patients with primary hypercholesterolemia significantly reduces cardiovascular morbidity and mortality.
There are conditions in which diet alone cannot control serum cholesterol levels, for example, patients with hereditary disorders attributable to defects in receptor mediated LDL catabolism (familial hypercholesterolemia), those with hypercholesterolemia due to overproduction of VLDL and LDL by the liver (familial combined hyperlipidemia), patients with secondary hypercholesterolemia due to the nephrotic syndrome, as well as many adult patients with type III hyperlipoproteinemia. The combination of a bile sequestrant with a drug that inhibits the hepatic synthesis of cholesterol, VLDL, or LDL has been used. However, this regimen unfortunately cannot be tolerated by all patients and is not consistently effective. The Consensus Conference on Cholesterol concluded that there was a need for newer hypocholesterolemic agents that are effective, safe and encourage patient compliance.
The HMGCoA reductase inhibitors, such as mevastatin and lovastatin, have provided a potential new mode of therapy for patients with significant hypercholesterolemia that does not respond adequately to diet therapy alone. These HMGCoA reductase inhibitors reduce the rate of formation of mevalonic acid which leads to a depletion of the cellular pool of cholesterol. This, in turn, results in an increased number of hepatic LDL receptors which yields a further decrease in serum cholesterol level (15). Drugs such as lovastatin seem to be relatively effective, particularly when used in combination with bile sequestering compounds such as cholestyramine, but they are not without side effects. Gastrointestinal disturbances, including flatulence, diarrhea, constipation and nausea are the most frequently reported side effects. Abdominal pain, cramps, insomnia, rash and headache are also reported. Paradoxically, increased levels of HMGCoA reductase have been reported in animals treated with high doses of mevinolin. While there is no published data to indicate that this will happen in humans there is the possibility that prolonged treatment of hypercholesterolemia with an HMGCoA reductase inhibitor will lead to increased enzyme levels and ultimately to resistance to the drug. Changes in liver function tests have also lead to withdrawal of patients from lovastatin treatment and there is some concern over the development of cataracts. The use of cholesterol-lowering drugs is projected to increase significantly, and thus there is a continuing need for safe and effective drugs.
Two of the colony-stimulating factors, CSF-1 and GM-CSF, have been observed to exert profound decreases in serum cholesterol levels (16, 17) equal to or greater than that observed with inhibitors of HMGCoA reductase like lovastatin. For GM-CSF average decreases of 37% were observed. Although the mechanism of these effects has yet to be determined, these factors could be potentially useful in the treatment of hypercholesterolemia and thus atherosclerosis.
There is increasing evidence that high levels of high density lipoproteins (HDL) are anti-atherogenic (18), probably due to their role in `reverse cholesterol transport` which mediates the movement of cholesterol from the peripheral tissues back to the liver. Proteins involved in this process include apolipoproteins AI and AII (18, 19), lecithin-cholesterol acyl-transferase (LCAT)(20), cholesteryl ester transfer protein (CETP)(21), and HDL receptors (22). These proteins are all potential targets for pharmacological intervention. Other proteins involved in cholesterol metabolism such as acetyl CoA-cholesterol acyl transferase (23), cholesterol 7.alpha.-hydroxylase (24), and AMP-activated protein kinase and kinase kinase (25) are also likely targets for future drugs.
4. HYPERTENSION AND ANTIHYPERTENSIVE DRUGS
Hypertension is another of the leading risk factors for atherosclerosis (26), although the molecular mechanisms involved in its stimulation of atherogenesis have yet to be elucidated. The increased expression of PDGF .alpha.-receptors but not PDGF a-receptors in arterial smooth muscle cells in response to hypertension (27), and the increased PDGF-B transcription in endothelial cells in response to increased shear forces (28) may be involved in this process. Atherosclerotic arteries have also been observed to be defective in EDRF (Endothelial-Derived Relaxation Factor) mediated vasodilation (29). Studies with specific inhibitors of EDRF synthesis have indicated that the entire circulation is in a constant state of vasodilation due to the continuous release of this factor from endothelial cells. Some antihypertensives (e.g. .beta.-blockers, nifedipine) have been demonstrated to be efficacious in preventing atherosclerosis from developing, but not necessarily in inducing regression of lesions that are already present.
Although treatment of high blood pressure has been successful in reducing the arteriolar complications such as brain hemorrhage from rupture of microaneurisms, lacunar strokes from occlusion of arterioles by fibrinoid necrosis or adrenal failure from hypertensive nephropathy, atherosclerotic complications such as myocardial infarction are not uniformly prevented by such treatment (26). Antihypertensive drugs have different effects on factors that might adversely affect atherosclerosis. For example, they can have adverse effects on lipoprotein profiles or hemodynamic factors that influence the occurrence of blood flow disturbances (26). It has been estimated that the magnitude of this adverse effect can be enough to completely offset the benefit of treating mild hypertension. Many antihypertensives also have other undesirable side effects. There is thus a continuing need for new antihypertensives for the treatment of cardiovascular disease.
The Calcitonin/Calcitonin gene-related peptide (CGRP) gene encodes the calcium lowering hormone Calcitonin (CCPI), the vascular relaxant neuropeptide, CGRP and a calcitonin carboxy-terminal peptide (CCPII) of unknown function (92).
The regulation of calcitonin gene expression is controlled at multiple levels. At the level of transcription, the expression of the calcitonin gene is enhanced by phorbol esters, cAMP analogues, 1,25-dihydroxy vitamin D.sub.3 and dexamethazone (97). Glucocortoids enhance calcitonin mRNA level while lowering CGRP mRNA.
CGRP is a potent endogenous vasodilator. The peptide is released from perivascular nerve endings and can normally be detected in the circulation. CGRP levels in patients with essential hypertension is lower than in normal subjects (98). The decrease of CGRP is closely related with the severity of hypertension. CGRP has also been shown to modulate the vasopressor effects of norepinephrine and angiotensin II (99)
5. STENOSIS AND RESTENOSIS
Invasive cardiovascular surgical procedures, such as percutaneous translumenal coronary angioplasty (PTCA, using a balloon catheter) and aorto-coronary bypass surgery (ACBS), that are currently employed in treating the coronary stenosis or occlusion caused by atherosclerosis represent a major therapeutic advance for managing coronary heart disease (CHD). However, the cellular proliferative response and associated intimal hyperplasia that can follow the damage to blood vessels that occurs with these procedures leads to complications which cannot be effectively controlled by presently available drugs, and can be more detrimental than the original condition. The development of these complications, termed restenosis (in the case of PTCA) or stenosis (in the case of ACBS), has similarities to the development of atherosclerosis. After vascular injury and the resulting de-endothelialization, one of the initial events in the development of stenosis is platelet adherence to the damaged area (30). There is good evidence from animal models that the accumulation of neointimal smooth muscle cells (SMC) which leads to restenosis is due to the chemotactic properties of platelet-derived PDGF (7, 31) (possibly via induction of a bFGF autocrine loop). Polyclonal antibodies specific for PDGF-AB inhibit restenosis. PDGF .beta.-receptors are upregulated in SMCs at the stage in restenosis when they are migrating from the media to the intima (32). Basic FGF is an effective mitogen for vascular smooth muscle cells in the medial layer of the artery wall (5), but once they have migrated to the intima the major mitogenic signal is, as in atherogenesis, unknown.
The potential benefit of having effective inhibitors of stenosis or restenosis is considerable since 190,000 coronary angioplasties and about 330,000 aorto-coronary bypass surgeries are carried out each year in the USA alone. Stenosis or restenosis is a severe problem in about a third of these procedures, often requiring multiple surgeries, which at $7,500 for angioplasty and $30,000-40,000 for bypass surgery is an expensive process. An effective antistenotic drug might be one that prevents platelet adhesion to the site of injury (e.g. by stimulating EDRF-dependent guanylate cyclase or down-regulating cell adhesion molecules), stimulates re-endothelial growth (e.g. by stimulating VEGF production by vascular smooth muscle cells) or inhibits smooth muscle cell migration (e.g. by down-regulating vascular smooth muscle cell PDGF receptors) or proliferation (e.g. by down-regulating vascular smooth muscle cell FGF receptors). A combination of such drugs would probably be most effective. Platelet inhibitors like aspirin, which can limit platelet aggregation but cannot inhibit platelet-endothelial adhesion, reduce restenosis rates slightly.
6. THROMBOSIS
It has been known for some time that thrombosis is just as important as atherogenesis in causing ischemic heart disease. Several studies have shown strong relationships between high levels of factor VII coagulant activity and fibrinogen, and subsequent risk of clinically manifest ischemic heart disease (33). In the prospective Northwick Park Heart Study high levels of fibrinogen were associated with mortality from all causes and the relationship between fibrinogen and incidence of coronary heart disease was stronger than that for cholesterol (34). High levels of fibrinogen are likely to predispose to thrombosis, since fibrinogen/fibrin are constituents of atherosclerotic plaques, fibrinogen concentration is an important determinant of blood viscosity, and elevated levels of fibrinogen lead to an increase in platelet aggregation. Regarding the role of factor VII in heart disease, it is probably also not a coincidence that factor VII is the only clotting factor zymogen which has some biological activity, all of the others requiring to be activated before they can exert any functional effect. High levels of plasminogen activator inhibitor (PAI-1) have also been associated with myocardial infarction (35). There is an extensive literature indicating the efficacy of antithrombolytic treatment (e.g. aspirin, tPA, streptokinase, nitroglycerin) in reducing both the morbidity and mortality due to angina and myocardial infarction.
There are several mechanisms by which atherosclerotic plaques can become thrombogenic. Many of the substances produced by cells at places can stimulate the production of activators of coagulation, or inhibitors of fibrinolysis. For example, IL-1 and TNF-.alpha. produced by macrophages can stimulate the endothelial cell surface accumulation of tissue factor. Rupture of plaques at an advanced stage of formation also results in the release of tissue factor. Macrophage or platelet derived TGF-.beta., or macrophage-derived IL-1 and TNF-.alpha. also stimulate the production of PAI-1 in endothelial cells (36). Increased PAI-1 synthesis is also stimulated in vascular smooth muscle cells by PDGF-B.beta. and TGF.beta. (37), which is particularly relevant to situations involving vascular injury where adhering platelets produce these factors.
7. TRANSCRIPTIONAL REGULATION--GENERAL
Pharmaceuticals which increase or decrease the expression of genes associated with cardiovascular diseases will have important clinical application for the treatment of a variety of diseases and conditions, including myocardial infarction, angina, stroke, hypertension, hypercholesterolemia, restenosis and diabetes. This invention includes a method for discovery of compounds which modulate the expression of these genes and describes the use of such compounds. The general approach is to screen compound libraries for substances which increase or decrease expression of genes associated with cardiovascular diseases.
Examples of such genes would include those for apolipoprotein (a), the protein component of the `atherogenic` Lipoprotein (a) which is specific to this particle (38); apolipoprotein AI, the major protein component of high density lipoproteins (HDL), the `antiatherogenic` lipoprotein component of serum (18); apolipoprotein B, the main protein component of LDL (39); LDL receptor, the major hepatic receptor for LDL which removes LDL from the circulation (40); scavenger receptor, a major receptor for the uptake of oxidized LDL by macrophages at atherosclerotic plaques (41); cholesterol 7.alpha.-hydroxylase, the rate-limiting enzyme in the hepatic conversion of cholesterol to bile acids, and thus its clearance from the body (24); vascular endothelial growth factor (VEGF), a potent and specific growth factor for endothelial cells, whose production by vascular smooth muscle cells may lead to repair of vascular damage (e.g. after angioplasty) (42); .beta.-fibrinogen, the major precursor protein involved in the formation of thrombi (43); colony stimulating factor-1, the major factor involved in stimulating viability, proliferation and differentiation of cells in the mononuclear phagocyte series (e.g. monocytes, macrophages) (41); and monocyte chemoattractant protein-1 (MCP-1), a protein produced by vascular endothelial and smooth muscle cells in response to a variety of stimuli (e.g. oxidized LDL, IL-1), and postulated to be involved in stimulating migration of monocytes/macrophages into atherosclerotic plaques (3). In order to reduce atherosclerosis, or restenosis after balloon angioplasty, and thus alleviate the symptoms of cardiovascular disease, one would search for compounds which down-regulate the expression of the genes for apolipoprotein (a), apolipoprotein B, MCP-1, CSF-1, .beta.-fibrinogen and scavenger receptor, and compounds which upregulate the expression of the genes for apolipoprotein AI, hepatic LDL receptor, cholesterol 7.alpha.-hydroxylase and VEGF.
Similarly, one would search for compounds which inhibit the expression of genes involved in the oxidation of LDL, such as 15-lipoxygenase (1); inhibit the expression of genes involved in smooth muscle cell proliferation or migration to the intima, such as PDGF or basic FGF (5, 7); inhibit the expression of genes involved in the biosynthesis of cholesterol such as HMGCoA reductase (25); inhibit the expression of cell adhesion molecule genes (e.g. VCAM-1 (2), vitronectin receptor) involved in adhesion of cells like macrophages and platelets to the endothelium; inhibit the expression of genes involved in formation or inhibition of the breakdown of thrombi, such as plasminogen activator inhibitor; inhibit the expression of genes whose inhibition would cause a reduction of hypertension, such as preprorenin or angiotensinogen (44).
The expression of a specific gene can be regulated at any step in the process of producing an active protein. Modulation of total protein activity may occur via transcriptional, transcript-processing, translational or post-translational mechanisms. Transcription may be modulated by altering the rate of transcriptional initiation or the progression of RNA polymerase (45). Transcript-processing may be influenced by circumstances such as the pattern of RNA splicing, the rate of mRNA transport to the cytoplasm or mRNA stability. This invention concerns the use of molecules which act by modulating the in vivo concentration of their target proteins via regulating gene transcription. The functional properties of these chemicals are distinct from previously described molecules which also affect gene transcription.
Researchers have documented the regulation of transcription in bacteria by low molecular weight chemicals (46, 47). Extracellular xenobiotics, amino acids and sugars have been reported to interact directly with an intracellular proteinaceous transcriptional activator or repressor to affect the transcription of specific genes.
Transcriptional regulation is sufficiently different between procaryotic and eucaryotic organisms so that a direct comparison cannot readily be made. Procaryotic cells lack a distinct membrane bound nuclear compartment. The structure and organization of procaryotic DNA elements responsible for initiation of transcription differ markedly from those of eucaryotic cells.
The eucaryotic transcriptional unit is much more complex than its procaryotic counterpart and consists of additional elements which are not found in bacteria. Eucaryotic transcriptional units include enhancers and other cis-acting DNA sequences (48, 49). Procaryotic transcription factors most commonly exhibit a "helix-turn-helix" motif in the DNA binding domain of the protein (50, 51). Eucaryotic transcriptional factors frequently contain a "zinc finger" (51, 52), "helix-loop-helix" or a "leucine zipper" (53) in addition to sometimes possessing the "helix-turn-helix" motif (54). Furthermore, several critical mechanisms at the post-transcriptional level such as RNA splicing and polyadenylation are not found in procaryotic systems (55, 56).
In higher eucaryotes, modulation of gene transcription in response to extracellular factors can be regulated in both a temporal and tissue specific manner (57). For example, extracellular factors can exert their effects by directly or indirectly activating or inhibiting transcription factors (57, 58).
Modulators of transcription factors involved in direct regulation of gene expression have been described, and include those extracellular chemicals entering the cell passively and binding with high affinity to their receptor-transcription factors. This class of direct transcriptional modulators include steroid hormones and their analogs, thyroid hormones, retinoic acid, vitamin and its derivatives, and dioxins, a chemical family of polycyclic aromatic hydrocarbons (52, 59, 60).
Dioxins are molecules generally known to modulate transcription, however, dioxins bind to naturally-occurring receptors which respond normally to xenobiotic agents via transcriptionally activating the expression of cytochrome P450, part of an enzyme involved in detoxification. Similarly, plants also have naturally occurring receptors to xenobiotics to induce defense pathways. For example, the fungal pathogen Phytophthora megasperma induces an anti-fungal compound in soybeans. Such molecules which bind to the defined ligand binding domains of such naturally occurring receptors are not included on the scope of this invention.
The clinical use of steroid hormones, thyroid hormones, vitamin D.sub.3 and their analogs demonstrates that agents which modulate gene transcription can be used for beneficial effects, although these agents can exhibit significant adverse side effects. Obviously, analogs of these agents could have similar clinical utility as their naturally occurring counterparts by binding to the same ligand binding domain of such receptors.
Indirect transcriptional regulation involves one or more signal transduction mechanisms. The regulation typically involves interaction with a trans-membrane signal transducing protein, the protein being part of a multistep intracellular signaling pathway, the pathway ultimately modulating the activity of nuclear transcription factors. This class of indirect transcriptional modulators include polypeptide growth factors such as platelet-derived growth factor, epidermal growth factor, cyclic nucleotide analogs, and mitogenic tumor promoters (61, 62, 63).
It is well documented that a large number of chemicals, both organic and inorganic, e.g. metal ions, can non-specifically modulate transcription.
Researchers have used nucleotide analogs in methods to modulate transcription. The mechanism involves incorporating nucleotide analogs into nascent mRNA or non-specifically blocking mRNA synthesis. Similarly, researchers have used alkylating agents, e.g. cyclophosphamide, or intercalating agents, e.g. doxorubicin, to non-specifically inhibit transcription.
Moreover, chemical inhibitors of hydroxymethyl-glutaryl CoA reductase, e.g. lovastatin, are known to modulate transcription by indirectly increasing expression of hepatic low density lipoprotein receptors as a consequence of lowered cholesterol levels.
The best examples of promoters subject to pharmaceutical intervention are provided by those of the steroid responsive genes. Nolvadex (Tamoxifen), for example, regulates the expression of estrogen responsive genes in the control of breast cancer, while Eulexin (flutamide) demonstrates dramatic palliative efficacy in the treatment of prostatic carcinoma by blocking the action of the testosterone receptor. Similarly, steroid receptor agonists are used as anti-inflammatory agents, contraceptives, etc. It is now becoming increasingly apparent that a number of drugs whose mechanism was previously unknown, also act by specifically modulating the expression of various target genes. For example, the immunosuppressive agents cyclosporin A and FK506, both inhibit the transcription of several genes involved in T cell activation, in particular interleukin 2 (64, 65). The cellular receptors for these compounds are the proteins termed cyclophilins, which may act, at least in part, by inhibiting the Ca.sup.2+ /calmodulin-dependent protein phosphatase calcineurin. Altered phosphatase activity presumably modulates gene expression by altering the phosphorylation state of specific transcription factors, or transcription factor activity modulators. The anti-inflammatory action of aspirin has also recently been shown to be at least partly due to altered levels of gene expression.
Signal effector type molecules such as cyclic AMP, diacylglycerol, and their analogs are known to regulate a broad spectrum of genes at the transcriptional level by acting as part of a multistep protein kinase cascade reaction. These signal effector type molecules bind to domains on proteins which are thus subject to normal physiological regulation by low molecular weight ligands (67, 68).
The specific use of sterol regulatory elements from the LDL receptor gene to control expression of a reporter gene has recently been documented in PCT/US88/10095. One aspect of PCT/US88/10095 deals with the use of specific sterol regulatory elements coupled to a reporter as a means to screen for drugs capable of stimulating cells to synthesize the LDL receptor. PCT/US88/10095 describes neither the concept of simultaneously screening large numbers of chemicals against multiple target genes nor the existence of transcriptional modulators which (a) do not naturally occur in the cell, (b) specifically transcriptionally modulate expression of the gene encoding the protein of interest associated with cardiovascular disease, and (c) directly bind to DNA or RNA, or directly bind to a protein through a domain of such protein which is not a defined ligand binding domain of a nuclear, transcriptionally activating receptor which naturally occurs in the cell, the binding of a ligand to which ligand binding domain is normally associated with the defined physiological effect. The main focus of PCT/US88/10095 is the use of the sterol regulatory elements from the LDL receptor as a means to inhibit expression of toxic recombinant biologicals.
The use of molecules to directly and specifically modulate transcription of genes associated with cardiovascular disease as described herein has not previously been reported and its use will be considered novel since available literature does not propose the use of a molecule, as described, in a method to specifically modulate transcription. Instead, the available literature has reported methods which define domains of transcriptional regulating elements of a gene.
Further, the practice of using a reporter gene to analyze nucleotide sequences which regulate transcription of a gene-of-interest is well documented. The demonstrated utility of a reporter gene is in its ability to define domains of transcriptional regulatory elements of a gene- of-interest. Reporter genes which express proteins, e.g. luciferase, are widely utilized in such studies. Luciferases expressed by the North American firefly, Photinus pyralis and the bacterium, Vibrio fischeri were first described as transcriptional reporters in 1985 (69, 70).
A method to define domains of transcriptional regulating elements of a gene-of-interest typically has also involved use of phorbol esters, cyclic nucleotide analogs, concanavalin A, or steroids, molecules which are commonly known as transcriptional modulators. However, available literature shows that researchers have not considered using a transcription screen to identify specific transcriptional modulators. Apparently, success would be unlikely in doing so, however, we have demonstrated previously that this is not the case.
There is utility in developing the method of transcriptional modulation of genes associated with cardiovascular disease by using such molecules as described herein. This method will allow the development of novel pharmaceuticals and circumvent many of the problems associated with the therapeutic use of recombinant biological factors where clinical use of protein factors is relevant.
Problems associated with the therapeutic use of recombinant biological factors include the technical difficulties of large scale protein purification, the high costs of protein production, the limited shelf-life of most proteins and in some cases a short biological half-life of the administered protein in the organism. Additionally, therapeutic delivery of proteins normally requires injection and frequently induces an immune reaction in situations where chronic administration is required. The method described herein provides a means of up-regulating the expression of proteins which are not readily amenable to administration as injectable biologicals.
Another potential advantage of transcriptional modulation over a biological would be the production of high localized concentrations of proteins. For example, a major application of vascular endothelial growth factor (VEGF) (42) as a biological would presumably be in wound healing. A major application in this area would be in the treatment of vascular wounding caused by balloon angioplasty or coronary bypass surgery. A small molecule drug upregulating VEGF gene expression would have advantages over the biological. Increased gene expression and secretion of VEGF by vascular smooth muscle cells which are present at the site of injury would lead to high local concentrations of VEGF. This would stimulate re-endothelialization, thus preventing platelet adhesion, and therefore stenosis or restenosis (43). Large doses of a biological would have to be given intravenously or intramuscularly to produce an effect similar to the locally produced paracrine-acting factor. There would thus be a greater likelihood of incurring side effects such as abnormal vascularization, or edema due to the vascular permeabilizing properties of VEGF.
Furthermore, chemical molecules specifically regulating the activity of one member of a group of closely related proteins are difficult to produce. Bioactive molecules, structurally related at the protein level, may possess distinct regulatory elements at the DNA level which control their expression. Thus, molecules such as the chemical transcriptional modulators defined herein can provide a greater opportunity for specifically modulating the activity of structurally related proteins. Finally, the molecules described herein may also serve to mimic normal physiological response mechanisms, typically involving the coordinated expression of one or more groups of functionally related genes. Therefore, determining whether a molecule can specifically transcriptionally modulate the expression of a gene involved in cardiovascular disease and the ultimate clinical use of the molecule provides a therapeutic advantage over the use of single recombinant biologicals, or drugs which bind directly to the final target protein encoded by the gene-of-interest.
There is considerable evidence from the literature to indicate that many of the genes involved in cardiovascular disease are regulated in vivo at the transcriptional level. For example modulation of the transcription of the gene coding for apolipoprotein AI can be achieved by thyroid hormone or cholesterol (71, 72). Similarly, LDL receptor gene expression can be modulated by cholesterol metabolites, cholesterol 7.alpha.-hydroxylase by bile acids (73), CSF-1 by gamma-interferon (74), MCP-1 by minimally-modified LDL, gamma-interferon, TNF-.alpha. or interleukin-1 (75), and .beta.-fibrinogen by glucocorticoids and interleukin-6 (76).